Detection of HPV-induced invasive cancers and their precursor lesions with invasive potential

ABSTRACT

The invention is in the field of medicine and is concerned with a molecular diagnostic marker for progression to invasiveness of HPV-induced premalignant lesions and future metastatic potential of HPV-induced premalignant lesions and carcinomas. In particular the present invention relates to the use of the TSLC1 gene as marker for progression to invasive cervical cancer and metastatic potential of cervical lesions. The invention provides methods for detecting HPV-induced invasive cancers and their precursor lesions associated with tumor suppressor lung cancer 1 (TSLC1), comprising contacting a target cellular component of a test cell with a reagent that detects TSLC1 and detecting a reduction in the TSLC1 as compared to that of a comparable normal cell. The invention also provides molecular diagnostic markers for premalignant cervical lesions with invasive potential associated with tumor suppressor lung cancer 1 (TSLC1) in cytologically abnormal cervical smears and/or biopsies.

FIELD OF THE INVENTION

The invention is in the field of medicine and is concerned with amolecular diagnostic marker for human papillomavirus (HPV)-inducedinvasive cancers and their precursor lesions with invasive potentialsuch as invasive cervical cancer, premalignant cervical lesions withinvasive potential and high-risk human papillomavirus (HPV)-inducednon-cervical invasive cancers. In particular the present inventionrelates to the use of the TSLC1 gene as marker for progression toinvasiveness of HPV-induced premalignant lesions and future metastaticpotential of HPV-induced premalignant lesions and carcinomas, allowingfor a better risk assessment and treatment option for the individualpatient.

BACKGROUND OF THE INVENTION

Cancer of the uterine cervix is the second most common cancer in womenworld-wide and is responsible for approximately 250,000 cancer deaths ayear.

Cervical cancer development is characterized by a sequence ofpremalignant lesions, so called cervical intraepithelial neoplasia(CIN), which are graded I to III, referring to mild dysplasia (CIN I),moderate dysplasia (CIN II) and severe dysplasia/carcinoma in situ (CINIII). CIN I is also referred to as low grade squamous intraepitheliallesion (LSIL) and CIN II and CIN III together as high grade squamousintraepithelial lesion (HSIL).

Over the past decade it has been well established that cervicalcarcinogenesis is initiated by an infection with high-risk humanpapillomavirus (HPV). Expression of the viral oncogenes E6 and E7, whichdisturb the p53 and Rb tumor suppressor pathways, respectively, has beenshown to be essential for both the onset of oncogenesis and themaintenance of a malignant phenotype. However, consistent with amultistep process of carcinogenesis, additional alterations in the hostcell genome are required for progression of an hr-HPV infected cell toan invasive carcinoma.

In line with multiple events underlying cervical carcinogenesis is theobservation that only a small proportion of women infected withhigh-risk HPV will develop high-grade premalignant cervical lesions (CINIII) or cervical cancer, and in most women with premalignant cervicallesions the lesions regress spontaneously.

However, at present no markers exist to predict which premalignantlesions will regress or ultimately progress to cervical cancer.Therefore, general medical practice comprises the treatment of all womenwith morphologically confirmed CIN II and CIN III, in order to preventthe development of cervical cancer. Consequently, many women areunnecessarily treated and unnecessarily worried.

SUMMARY OF THE INVENTION

It has now surprisingly been found that the tumor suppressor lung cancer1 gene (TSLC1) is involved as a tumor suppressor gene in cervicalcarcinogenesis and that TSLC1 silencing, or a low level of expression ofthe TSLC1 gene, is an important determinant of cervical carcinogenesis.Compared to its role in the development of a subset of lung carcinomas,the silencing of TSLC1 plays an even more predominant role in cervicalcarcinogenesis. TSLC1 and the gene products thereof thus providevaluable molecular markers to diagnose invasive cervical lesions and/orto (better) predict which premalignant lesions have a high chance ofprogressing to cervical cancer.

Cervical cancer is almost exclusively associated with humanpapillomavirus (HPV) infection. Human papillomaviruses, constitute agroup of more than 100 types of viruses, as identified by variations inDNA sequence. The various HPVs cause a variety of cutaneous and mucosaldiseases. Certain types may cause warts, or papillomas, which are benign(noncancerous) tumors. Others have been found to cause invasivecarcinoma of the uterine cervix.

HPVs are broadly classified into low-risk and high-risk types, based ontheir ability to induce malignant changes in infected cells. Low riskHPV types such as 1, 2, 4, 6, 11, 13 and 32 are primarily associatedwith benign lesions or common warts while the high risk types, such as16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82 areprimarily associated with premalignant and malignant epithelial lesions.These high-risk types of HPV cause growths that are usually flat andnearly invisible, as compared with the warts caused by low-risk types,e.g. HPV-6 and HPV-11.

Therefore, the present invention is not only suited to detect invasivecervical cancer associated with tumor suppressor lung cancer 1 (TSLC1),but also other invasive cancers that are induced by HPV, particularly ofthe high-risk type and provides a method for the risk assessment ofpremalignant lesions becoming invasive.

Accordingly, the present invention provides methods of detectingHPV-induced invasive cancers and their precursor lesions associated withtumor suppressor lung cancer 1 (TSLC1) in a subject in need thereof,said method comprising contacting a cell component of a test cell of thesubject with a reagent that detects the level of the cell component inthe test cell and determining a modification in the level of the cellcomponent in the test cell as compared with a comparable healthy cell,wherein the cell component indicates the level of TSLC1 in the cell andthe modification indicates the presence of HPV-induced invasive cancer.

Very suitable HPV-induced invasive cancers or precursor lesions thereofin the context of the present invention are invasive cervical cancersand premalignant cervical lesions with invasive potential, but alsoinvasive cancers and premalignant lesions that are induced by HPV inother tissues such as from oral cavity, oropharynx, anus, rectum, penis,vulva, etc.

A test cell may be a neoplastic cell, a proliferating cervical cell, orany other cell wherein the presence of an HPV-induced invasive cancer orprecursor lesion thereof associated with tumor suppressor lung cancer 1is to be detected.

In another embodiment, the present invention provides methods ofdetecting HPV-induced invasive cancers and their precursor lesionsassociated with tumor suppressor lung cancer 1 (TSLC1) in a subject inneed thereof, said method comprising contacting a target cellularcomponent of a test cell with a reagent that detects TSLC1 and detectinga reduction in the TSLC1 as compared to that of a comparable normalcell. Preferably in said detection an increased methylation of the TSLC1promoter in the test cell and/or a reduced production of TSLC1 in thetest cell as compared to the comparable normal cell is determined.

In yet another embodiment, the present invention provides methods oftreating high-risk HPV-induced invasive cancers and their precursorlesions associated with modification of TSLC1 production in test cellsin a subject in need thereof, said method comprising contacting cells ofa patient suffering from said cancer with a therapeutically effectiveamount of a reagent that increases TSLC1 level in test cells.

In another aspect, the present invention relates to the use of moleculardiagnostic markers for the detection of HPV-induced invasive cancers andtheir precursor lesions associated with tumor suppressor lung cancer 1(TSLC1), wherein said marker indicates TSLC1 promoter methylation,expression of mRNA associated with production of TSLC1 polypeptideand/or allelic loss of the 11q23 chromosome. By such use, the risk ofprogression to invasive cancer may be determined.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the 5′ regulatory region (−895 to −1) and coding andtranscribed 3′ non-coding sequences to 1456) (SEQ ID NO:1), derived fromaccession start and stop codons are represented in italics and areunderlined.

DETAILED DESCRIPTION OF THE INVENTION

“Expression” refers to the transcription of a gene into structural RNA(rRNA, tRNA) or messenger RNA (mRNA) and, if applicable, subsequenttranslation into a protein.

The term “HPV-induced invasive cancer” refers to a carcinoma induced byhigh-risk HPV, which invades surrounding tissue.

The term “invasive cervical cancer” refers to a cervical carcinomainvading surrounding tissue.

The terms “premalignant lesion” and “precursor lesion” refer to a stagein the multistep cellular evolution to cancer with a strongly increasedchance to progress to a carcinoma. With classical morphology thepathologist is unable to predict in the individual patient which ofthese lesions will progress or regress. The current patent applicationrefers to a method, which can predict the progression to invasivecancer.

The term “invasive potential” refers to the potential to invadesurrounding tissue and consequently to become malignant.

The term “premalignant cervical lesion” refers to a stage in themultistep cellular evolution to cervical cancer with a stronglyincreased chance to progress to a cervical carcinoma. With classicalmorphology the pathologist is unable to predict in the individualpatient which of these lesions will progress or regress.

The term “capable of specifically hybridizing to” refers to a nucleicacid sequence capable of specific base-pairing with a complementarynucleic acid sequence and binding thereto to form a nucleic acid duplex.

A “complement” or “complementary sequence” is a sequence of nucleotideswhich forms a hydrogen-bonded duplex with another sequence ofnucleotides according to Watson-Crick base-paring rules. For example,the complementary base sequence for 5′-AAGGCT-3′ is 3′-TTCCGA-5′.

The term “stringent hybridization conditions” refers to hybridizationconditions that affect the stability of hybrids, e.g., temperature, saltconcentration, pH, formamide concentration and the like. Theseconditions are empirically optimised to maximize specific binding andminimize non-specific binding of the primer or the probe to its targetnucleic acid sequence. The terms as used include reference to conditionsunder which a probe or primer will hybridise to its target sequence, toa detectably greater degree than other sequences (e.g. at least 2-foldover background). Stringent conditions are sequence dependent and willbe different in different circumstances. Longer sequences hybridisespecifically at higher temperatures. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence at a defined ionic strength and pH. The T_(m)is the temperature (under defined ionic strength and pH) at which 50% ofa complementary target sequence hybridises to a perfectly matched probeor primer. Typically, stringent conditions will be those in which thesalt concentration is less than about 1.0 M Na ion, typically about 0.01to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes or primers (e.g.10 to 50 nucleotides) and at least about 60° C. for long probes orprimers (e.g. greater than 50 nucleotides). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. Exemplary low stringent conditions or “conditions of reducedstringency” include hybridization with a buffer solution of 30%formamide, 1 M NaCl, 1% SDS at 37° C. and a wash in 2×SSC at 40° C.Exemplary high stringency conditions include hybridization in 50%formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60° C.Hybridization procedures are well known in the art and are described ine.g. Ausubel et al, Current Protocols in Molecular Biology, John Wiley &Sons Inc., 1994.

The term “oligonucleotide” refers to a short sequence of nucleotidemonomers (usually 6 to 100 nucleotides) joined by phosphorous linkages(e.g., phosphodiester, alkyl and aryl-phosphate, phosphorothioate), ornon-phosphorous linkages (e.g., peptide, sulfamate and others). Anoligonucleotide may contain modified nucleotides having modified bases(e.g., 5-methyl cytosine) and modified sugar groups (e.g., 2′-O-methylribosyl, 2′-O-methoxyethyl ribosyl, 2′-fluoro ribosyl, 2′-amino ribosyl,and the like). Oligonucleotides may be naturally-occurring or syntheticmolecules of double- and single-stranded DNA and double- andsingle-stranded RNA with circular, branched or linear shapes andoptionally including domains capable of forming stable secondarystructures (e.g., stem-and-loop and loop-stem-loop structures).

The term “primer” as used herein refers to an oligonucleotide which iscapable of annealing to the amplification target allowing a DNApolymerase to attach thereby serving as a point of initiation of DNAsynthesis when placed under conditions in which synthesis of primerextension product which is complementary to a nucleic acid strand isinduced, i.e., in the presence of nucleotides and an agent forpolymerization such as DNA polymerase, and at a suitable temperature andpH. The (amplification) primer is preferably single stranded for maximumefficiency in amplification. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime thesynthesis of extension products in the presence of the agent forpolymerization. The exact lengths of the primers will depend on manyfactors, including temperature and source of primer. A “pair ofbi-directional primers” as used herein refers to one forward and onereverse primer as commonly used in the art of DNA amplification such asin PCR amplification.

The term “probe” refers to a single-stranded oligonucleotide sequencethat will recognize and form a hydrogen-bonded duplex with acomplementary sequence in a target nucleic acid sequence analyte or itscDNA derivative.

The TSLC1 (tumor suppressor in lung cancer 1) gene (Genbank AccessionNM_(—)014333) has originally been identified as a tumor suppressor genein the lung cancer cell line A549 by functional complementation studies(Kuramochi et al., 2001; Nature Genet 27, 427-430). It was shown thatre-expression of TSLC1 in the lung cancer cell line A549 suppressedtumorigenicity in nude mice. Moreover, loss or suppression of TSLC1expression in other lung cancer cell lines was shown to be correlated toboth tumorigenicity and spleen to liver metastasis in nude mice. TSLC1mRNA suppression in these cell lines was found to correlate tohypermethylation of the TSLC1 promoter.

Subsequent analysis of non-small cell lung cancers (NSCLC) revealedallelic loss at 11q23.2, the TSLC1 locus, in about 40% of lung cancersand within this group of tumors with allelic loss promoterhypermethylation of the other allele could be detected in 85% of cases.

TSLC1 encodes a member of the immunoglobulin super family of celladhesion molecules (IgCAMs), which consists of a wide variety ofcell-surface molecules that are characterized by an immunoglobulin unit.The TSLC1 protein is an N-linked glycoprotein of 75 kDa, which islocalized at the cell membrane and is involved in intracellular adhesionthrough homophilic trans-interaction (Masuda et al, 2002; J Biol Chem277, 31014-31019).

The present inventors have now established that TSLC1 silencing is afrequent event in cervical cancer cell lines. TSLC1 silencing is foundto result from TSLC1 promoter hypermethylation either or in not incombination with allelic loss. In vitro studies revealed a functionalinvolvement of TSLC1 inactivation in both anchorage independent growthand tumorigenicity of cervical cancer cells, whereas immortality andproliferation were not affected. This points to a role for TSLC1silencing in tumor invasion rather than proliferation. Loss of aputative tumor suppressor gene on chromosome 11 was already known to beinvolved in tumorigenicity of SiHa cervical cancer cells, and allelicloss at 11q22-23 has frequently been detected in invasive cervicalcarcinoma. These results indicate that inactivation of TSLC1 at 11q23.2might play a crucial role in cervical cancer invasion.

Analysis of cervical tissue specimens revealed that TSLC1 promoterhypermethylation is limited to only a subset of high-grade CIN lesionsbut detectable in up to 58% of invasive cervical carcinomas. Moreover,TSLC1 promoter hypermethylation can specifically be detected in cervicalsmears derived from women with cervical cancer. From archival smearstaken several years prior to cervical cancer diagnosis it is known, thatTSLC1 promoter hypermethylation may be detected long time before theappearance of carcinomas. Promoter hypermethylation may occur e.g.already seven years prior to evolvement of invasive carcinomas. Howeverthe time span may vary in individual cases so that detectablehypermethylation of the TSLC1 promoter may be present from 20 years oreven more to 1 year or less before the evolvement of invasive carcinoma.Analysis of cervical cancer cell lines revealed that silencing of TSLC1expression is present in as much as 91% (10/11) of cell lines. TSLC1silencing appeared not only associated with promoter methylation, butalso with allelic loss at the TSLC1 locus and yet unknown events,suggesting that the percentage of cervical cancers showing TSLC1promoter methylation is even an underestimation of the actual percentageof cases in which the TSLC1 gene is silenced.

Cervical cancer development is the ultimate result from an HPV infectionand is as such etiologically different from the tumor types for whichTSLC1 silencing has been described as a molecular marker (patent WO02/14557), i.e. lung, prostate, pancreatic and hepatocellular cancer,all of which are unrelated to HPV. Moreover, our data are in favour ofTSLC1 silencing providing a marker of progression to invasiveness ratherthan a proliferation marker as indicated in WO02/14557. Since TSLC1promoter methylation could also be demonstrated in cervical smears,which are widely used in screening and triage settings, the potentialimpact of TSLC1 silencing as marker of progression to invasiveness andmetastatic potential goes far beyond the level of tissue specimens.

Furthermore, whereas TSLC1 silencing provides a marker for theprogression of an HPV infected lesion to invasiveness and metastaticpotential of an HPV-infected lesion, the detection of specific HPVexpression patterns as described in WO 99/29890 is not necessarilyrelated to invasion and consequently represents a marker with a lowerspecificity of invasive cervical cancer.

Accordingly, the present invention provides methods of detectingHPV-induced invasive cancers and their precursor lesions associated withtumor suppressor lung cancer 1 (TSLC1) in a subject in need thereof, orindicative thereof, said method comprising contacting a cell componentof a test cell of the subject with a reagent that detects the level ofthe cell component in the test cell and determining a modification inthe level of the cell component in the test cell as compared with acomparable healthy cell, wherein the cell component indicates the levelof TSLC1 in the cell and the modification indicates the presence ofHPV-induced invasive cancers and their precursor lesions.

The present invention furthermore provides a method for detection of thepresence or absence of cells in an individual that have the potential toevolve to invasive cervical carcinoma although those cells are notdetectable as a lesion or precursor thereof by conventional means.

The present invention furthermore provides a method for assessment ofprognosis for individuals undergoing cervical cancer screening allowingfor stratification of future diagnostic follow-up and/or treatment ofthe individuals depending on the expression status of TSLC1 and/orhypermethylation status of the TSLC1 promoter, wherein individualsshowing reduced TSLC1 expression compared to healthy individuals orhypermethylation of TSLC1 promoter are subjected to diagnostic follow-upprocedures and/or testing intervals, or special treatment procedures.

The test cell of the subject may comprise a cell from a sample of skincells (e.g. in the case of warts, veruccas and the like, presumablycaused by cutaneous HPV infections), a sample of mucosal cells, such ascervical cells, and also other tissue such as oral cavity, oropharynx,penis, vulva, anus, rectum and other tissues wherein a cancer associatedwith HPV is to be detected. All such samples may be used as a sample ina method of the present invention. Preferably, a sample of a patient'scells comprise cervical cells as test cells. The cervical cells may e.g.be presented as a histological or cytological specimen. Cytologicalspecimens comprise conventional cervical smears as well as thin layerpreparations of cervical specimens.

A method of the present invention is particularly suited for thedetection of HPV-induced invasive cancers and their precursor lesionsassociated with tumor suppressor lung cancer 1 (TSLC1) that are inducedby high-risk HPVs. A method of detecting HPV-induced invasive cancersand their precursor lesions associated with tumor suppressor lung cancer1 (TSLC1) may accordingly relate to the measurement of TSLC1 expression,such as in the form of measuring TSLC1 gene transcripts and/orsubsequent proteins translated from said transcripts. Also a method ofdetecting HPV-induced invasive cancers and their precursor lesions maycomprise measuring TSLC1 promoter methylation as an indication of TSLC1expression capacity and/or TSLC1 protein production capacity.

FIG. 1 shows the cg-rich promoter region upstream of the atg start codonin the TSLC1 gene and the coding region for the TSLC1 protein.Methylation of the cg-rich promoter region will result in a sharplydecreased transcription or even complete blockage of transcription.Therefore, the promoter region provides a positive marker sequence forthe expression potential of this gene. Alternatively, the expression ofthe TSLC1 gene may be detected by measuring gene transcripts. As such,the coding region for the TSLC1 protein in this gene provides a markersequence for detection of transcripts of the gene. In yet anotheralternative, the expression of the TSLC1 gene may be detected bymeasuring TSLC1 protein directly.

The test cell component contacted can thus be nucleic acid, such as DNAor RNA, preferably mRNA, or protein. When a cell component is protein,the reagent is typically an anti-TSLC1 antibody. When the component isnucleic acid, the reagent is typically a nucleic acid (DNA or RNA) probeor (PCR) primer. By using such probes or primers, gene expressionproducts, such as mRNA may for example be detected. Alternatively, whenthe component is nucleic acid, the reagent may also be a restrictionendonuclease, preferably a methylation sensitive restrictionendonuclease for the detection of the presence of methyl groups on thetest cell nucleic acid, said test cell nucleic acid then preferablybeing DNA.

The test cell component may be detected directly in situ or it may beisolated from other cell components by common methods known to those ofskill in the art before contacting with the reagent (see for example,“Current Protocols in Molecular Biology”, Ausubel et al. 1995. 4thedition, John Wiley and Sons; “A Laboratory Guide to RNA: Isolation,analysis, and synthesis”, Krieg (ed.), 1996, Wiley-Liss; “MolecularCloning: A laboratory manual”, J. Sambrook, E. F. Fritsch. 1989. 3 Vols,2nd edition, Cold Spring Harbor Laboratory Press)

Detection methods include such analyses as Southern and Northern blotanalyses, RNase protection, immunoassays, in situ hybridization, PCR(Mullis 1987, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), LCR(Barany 1991, Proc. Natl. Acad. Sci. USA 88:189-193; EP Application No.,320,308), 3SR (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA87:1874-1878), SDA (U.S. Pat. Nos. 5,270,184, and 5,455,166), TAS (Kwohet al., Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi et al., 1988, Bio/Technology 6:1197), Rolling CircleAmplication (RCA) or other methods for the amplification of DNA. In analternative method RNA may be detected by such methods as NASBA (L.Malek et al., 1994, Meth. Molec. Biol. 28, Ch. 36, Isaac P G, ed.,Humana Press, Inc., Totowa, N.J.) or TMA.

Nucleic acid probes, primers and antibodies can be detectably labeled,for instance, with a radioisotope, a fluorescent compound, abioluminescent compound, a chemiluminescent compound, a metal chelator,an enzyme or a biologically relevant binding structure such as biotin ordigoxygenin. Those of ordinary skill in the art will know of othersuitable labels for binding to the reagents or will be able to ascertainsuch, using routine experimentation.

Other methods for detection include such analyses as can be performedwith nucleic acid arrays (See i.a. Chee et al., 1996, Science274(5287):610-614). For example, DNA arrays may be used for thedetection of nucleic acids according to the invention. Such arrayscomprise oligonucleotides with sequences capable of hybridizing understringent conditions to the nucleic acid cell component of which thelevel is detected in a method of the present invention.

Since the present invention shows that a decreased level of TSLC1transcription is often the result of hypermethylation of the TSLC1 gene,it is often desirable to directly determine whether the TSLC1 gene ishypermethylated. In particular, the cytosine rich areas termed “CpGislands”, which lie in the 5′ regulatory regions of genes are normallyunmethylated. The term “hypermethylation” includes any methylation ofcytosine at a position that is normally unmethylated in the TSLC1 genesequence (e.g. the TSLC1 promoter). Hypermethylation can for instance bedetected by restriction endonuclease treatment of the TSLC1polynucleotide (gene) and Southern blot analysis. Therefore, in aninvention method wherein the cellular component detected is DNA,restriction endonuclease analysis is preferred to detecthypermethylation of the TSLC1 gene. Any restriction endonuclease thatincludes CG as part of its recognition site and that is inhibited whenthe C is methylated, can be utilized. Methylation sensitive restrictionendonucleases such as BssHII, MspI, NotI or HpaII, used alone or incombination, are examples of such endonucleases. Other methylationsensitive restriction endonucleases will be known to those of skill inthe art.

Other methods for the detection of TSLC1 promoter hypermethylationinvolve bisulfite modification of DNA, in which the unmethylatedcytosines are converted to an uracil whereas the methylated cytosinesare protected from chemical modification. Subsequent PCR amplificationand sequencing will reveal whether cytosines in CpG islands aremaintained in case of methylation or replaced by a uracil in case of anunmethylated status. Another method involves the treatment a PCRamplified product generated from bisulfite modified DNA with restrictionendonuclease that includes CG as part of its recognition site.

An alternative means to test for methylated sequences is a methylationspecific PCR, which is also based on bisulfite modification of DNA,followed by specific PCR reactions that target CpG rich sequences.

For purposes of the invention, an antibody (i.e., an anti-TSLC1antibody) or nucleic acid probe specific for TSLC1 may be used to detectthe presence of TSLC1 polypeptide (using antibody) or TSLC1polynucleotide (using nucleic acid probe) in biological fluids ortissues. Oligonucleotide primers based on any coding sequence region andregulatory sequence region in the TSLC1 sequence are useful foramplifying DNA, for example by PCR.

When using PCR primers, nucleic acid probes or restrictionendonucleases, the 5′ regulatory region and coding sequence of the TSLC1sequence is analysed.

Any specimen containing a detectable amount of TSLC1 polynucleotide orTSLC1 polypeptide antigen can be used. Nucleic acid can also be analyzedby RNA in situ methods that are known to those of skill in the art suchas by in situ hybridization. Preferred samples for testing according tomethods of the invention include such specimens as (cervical) smearsand/or (cervical) biopsies and the like. Preferably, cytologicalabnormal (cervical) smears and/or biopsies of high-grade (pre)malignantlesions are used as samples for testing. Although the subject can be anymammal, preferably the subject is human.

The invention methods can utilize antibodies immunoreactive with TSLC1polypeptide, the predicted amino acid sequence of which is available asGenBank Accession No. BAA75822, or immunoreactive fragments thereof. Anantibody preparation that consists essentially of pooled monoclonalantibodies with different epitopic specificities, as well as distinctmonoclonal antibody preparations can be used. Monoclonal antibodies aremade against antigen containing fragments of the protein by methods wellknown to those skilled in the art (Kohler, et al., Nature, 256:495,1975).

The term antibody as used in this invention is meant to include intactmolecules as well as fragments thereof, such as Fab and F(ab′)2, whichare capable of binding an epitopic determinant on TSLC1. Antibody asused herein shall also refer to other protein or non-protein moleculeswith antigen binding specificity such as miniantibodies,peptidomimetics, anticalins etc.

Monoclonal antibodies can be used in the invention diagnostic methods,for example, in immunoassays in which they can be utilized in liquidphase or bound to a solid phase carrier. In addition, the monoclonalantibodies in these immunoassays can be detectably labelled in variousways. Examples of types of immunoassays that can utilize monoclonalantibodies of the invention are competitive and non-competitiveimmunoassays in either a direct or indirect format. Examples of suchimmunoassays are the radioimmunoassay (RIA) and the sandwich(immunometric) assay. Detection of the antigens using the monoclonalantibodies of the invention can be done utilizing immunoassays that arerun in either the forward, reverse, or simultaneous modes, includingimmunohistochemical or immunocytochemical assays on physiologicalsamples. Those of skill in the art will know, or can readily discern,other immunoassay formats without undue experimentation.

Monoclonal antibodies can be bound to many different carriers and usedto detect the presence of TSLC1. Examples of well-known carriers includeglass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, agaroses andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of the invention. Those skilled in the art will know ofother suitable carriers for binding monoclonal antibodies, or will beable to ascertain such using routine experimentation.

In performing the assays it may be desirable to include certain“blockers” in the incubation medium (usually added with the labeledsoluble antibody). The “blockers” are added to assure that non-specificproteins, proteases, or antiheterophilic immunoglobulins to anti-TSLC1immunoglobulins present in the experimental sample do not cross-link ordestroy the antibodies on the solid phase support, or the radiolabelledindicator antibody, to yield false positive or false negative results.The selection of “blockers” therefore may add substantially to thespecificity of the assays described in the present invention. A numberof nonrelevant (i.e., nonspecific) antibodies of the same class orsubclass (isotype) as those used in the assays (e.g., IgGl, IgG2a, IgM,etc.) can be used as “blockers”. The concentration of the “blockers”(normally 1-100 μg/μL) may be important, in order to maintain the propersensitivity yet to inhibit any unwanted interference by mutuallyoccurring cross-reactive proteins in the specimen.

In using a monoclonal antibody for the in vivo detection of antigen, thedetectably labeled monoclonal antibody is given in a dose that isdiagnostically effective. The term “diagnostically effective” means thatthe amount of detectably labeled monoclonal antibody is administered insufficient quantity to enable detection of the site having the TSLC1antigen for which the monoclonal antibodies are specific. Theconcentration of detectably labelled monoclonal antibody which isadministered should be sufficient such that the binding to those cellshaving TSLC1 is detectable compared to the background, depending uponthe in vivo imaging or detection method employed, such as MRI, CAT scan,and the like. Further, it is desirable that the detectably labelledmonoclonal antibody be rapidly cleared from the circulatory system inorder to give the best target-to-background signal ratio.

As a rule, the dosage of detectably labelled monoclonal antibody for invivo diagnosis will vary depending on such factors as age, sex, andextent of disease of the individual. The dosage of monoclonal antibodycan vary from about 0.001 mg/m² tumor surface to about 500 mg/m²,preferably 0.1 mg/m² to about 200 mg/m², most preferably about 0.1 mg/m²to about 10 mg/m². Such dosages may vary, for example, depending onwhether multiple injections are given, tumor burden, and other factorsknown to those of skill in the art.

For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay that is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that the half-life of theradioisotope be long enough so that it is still detectable at the timeof maximum uptake by the target, but short enough so that deleteriousradiation with respect to the host is minimized. Ideally, a radioisotopeused for in vivo imaging will lack a particle emission, but produce alarge number of photons in the 140-250 keV range, which may be readilydetected by conventional gamma cameras.

For in vivo diagnosis, radioisotopes can be bound to immunoglobulineither directly or indirectly by using an intermediate functional group.Intermediate functional groups which often are used to bindradioisotopes which exist as metallic ions to immunoglobulins are thebifunctional chelating agents such as diethylenetriaminepentacetic acid(DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules.Typical examples of metallic ions that can be bound to the monoclonalantibodies of the invention are ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr, and²⁰¹Tl.

A monoclonal antibody useful in the invention methods can also belabeled with a paramagnetic isotope for purposes of in vivo diagnosis,as in magnetic resonance imaging (MRI) or electron spin resonance (ESR).In general, any conventional method for visualizing diagnostic imagingcan be utilized. Usually gamma and positron emitting radioisotopes areused for camera imaging and paramagnetic isotopes for MRI. Elements thatare particularly useful in such techniques include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy,⁵²Cr and ⁵⁶Fe.

Other, e.g. ex vivo, diagnostic methods for the detection of TSLC1production, TSLC1 gene expression or disorders therein, include methodswherein a sample for testing is provided, which sample comprises a cellpreparation from cervical or other tissue. Preferably such samples areprovided as smears. In order to provide for efficient testing schemes,cytologically abnormal (cervical) smears and/or biopsies of high-grade(pre)malignant lesions are used as samples for testing.

A cell or tissue sample obtained from a mammal, preferably a human, issuitably pretreated to allow contact between a target cellular componentof a test cell comprised in said sample with a reagent that detectsTSLC1 and detecting a reduction in the TSLC1 as compared to that of acomparable normal cell. Samples may be mounted on a suitable support toallow observation of individual cells. Examples of well-known supportmaterials include glass, polystyrene, polypropylene, polyethylene,polycarbonate, polyurethane, optionally provided with layers to improvecell adhesion and immobilization of the sample, such as layers ofpoly-L-lysine or silane. Cervical smears or biopsies may for instance beprepared as for the Papanicolaou (Pap) test or any suitable modificationthereof as known by the skilled person, and may be fixed by proceduresthat allow proper access of the reagent to the target component. Incertain embodiments of the invention the cytological specimens areprovided as conventional smear samples or thin layer preparations ofcervical cells or any other kind of preparation known to those of skillin the art. If storage is required, routine procedures use bufferedformalin for fixation followed by paraffin embedding, which provides fora well-preserved tissue infrastructure. In order to allow forimmunohistochemical or immunofluorescent staining, the antigenicity ofthe sample material must be retrieved or unmasked. One method ofretrieving the antigenicity of formaldehyde cross-linked proteinsinvolves the treatment of the sample with proteolytic enzymes. Thismethod results in a (partial) digest of the material and mere fragmentsof the original proteins can be accessed by antibodies.

Another method for retrieving the immunoreactivity of formaldehydecross-linked antigens involves the thermal processing using heat or highenergy treatment of the samples. Such a method is described in e.g. U.S.Pat. No. 5,244,787. Yet another method for retrieving antigens fromformaldehyde-fixed tissues is the use of a pressure cooker (e.g.2100-Retriever), either in combination with a microwave or in the formof an autoclave, such as described in e.g. Norton, 1994. J. Pathol.173(4):371-9 and Taylor et al. 1996. Biotech Histochem 71(5):263-70.

Several alternatives to formaldehyde may be used, such as ethanol,methanol, methacarn or glyoxal, citrated acetone, or fixatives may beused in combination. Alternatively, the sample may be air-dried beforefurther processing.

In order to allow for a detection with nucleic acid probes, the samplematerial must be retrieved or unmasked in case of formalin fixed andparaffin embedded material. One method involves the treatment withproteolytic enzymes and a postfixation with paraformaldehyde.Proteolytic digestion may be preceded by a denaturation step in HCl.This method results in a (partial) digest of the material allowing theentry of probes to the target. No specific unmasking procedures arerequired in case of non-formalin fixed material, e.g. frozen material.Prior to hybridisation samples can be acetylated by treatment withtriethanolamine buffer.

The nucleic acid probes or antibodies are then contacted with the samplematerial in a suitable buffer and permitted to specifically hybridize orbind to their nucleic acid or protein target. Upon specific binding ofthe nucleic acid probes or antibodies to the target components, labeledprobes and/or antibodies may be detected by such methods as confocallaser scanning microscopy, bright field microscopy, flow cytometryoptionally in combination with fluorescence associated cell sorting, ormodifications of these techniques, which are well known to the personskilled in the art.

In one embodiment of a method of the invention an increased methylationof the TSLC1 promoter in the test cell and/or reduced production ofTSLC1 in the test cell is detected as compared to the comparable normalcell.

The present invention also provides methods for treating a subject withHPV-induced invasive cancers associated with modification of TSLC1production, or indicative thereof, comprising administering to a subjectwith the cancer a therapeutically effective amount of a reagent thatincreases TSLC1 expression. In HPV-induced invasive cancers associatedwith tumor suppressor lung cancer 1 (TSLC1), the TSLC1 nucleotidesequence is underexpressed as compared to expression in a normal cell,therefore, it is possible to design appropriate therapeutic ordiagnostic techniques directed to this sequence. Thus, nucleic acidsequences that increase TSLC1 expression at the transcriptional ortranslational level can be used and, for example, nucleic acid sequencesencoding TSLC1 (sense) could be administered to the subject with theHPV-induced invasive cancer, such as invasive cervical cancer.

The term “invasive cervical cancer” denotes malignant as well aspremalignant cell populations, which often appear to differ from thesurrounding tissue both morphologically and genotypically. Thesedisorders are found to be associated with absence of or reducedexpression of TSLC1. Essentially, any test cell abnormality that isetiologically linked to expression of TSLC1 could be consideredsusceptible to treatment using methods of the present invention thatemploy a reagent to increase TSLC1 expression.

Increased TSLC1 expression may be attained by suppression of methylationof TSLC1 polynucleotide when TSLC1 is under-expressed. When, upondiagnosis according to the present invention, the HPV-induced cancerdetected is associated with TSLC1 expression, such methylationsuppressive reagents as 5-azacytadine can be introduced to a cell.Alternatively, when, upon diagnosis according to the present invention,the invasive cervical cancer detected is associated withunder-expression of TSLC1 polypeptide, a sense polynucleotide sequence(the DNA coding strand) encoding TSLC1 polypeptide, or 5′ regulatorynucleotide sequences (i.e., promoter) of TSLC1 in operable linkage withTSLC1 polynucleotide can be introduced into the cell. Demethylases knownin the art could also be used to remove methylation.

The present invention also provides gene therapy for the treatment ofHPV-induced cancer associated with modification of TSLC1 production.Such therapy would achieve its therapeutic effect by introduction of theappropriate TSLC1 polynucleotide that contains a TSLC1 structural gene(sense), into cells of subjects having HPV-induced cancer. Schemes andprocedures for effecting gene therapeutic treatment are known in theart, e.g. in WO 02/14557. Delivery of sense TSLC1 polynucleotideconstructs can be achieved by using an expression vector or by using acolloidal dispersion system, preferably a recombinant expression vector,said recombinant expression vector preferably being a plasmid, a viralparticle or a phage.

The polynucleotide sequences used in the methods of the invention may bethe native, unmethylated sequence or, alternatively, may be a sequencein which a nonmethylatable analog is substituted within the sequence.Preferably, the analog is a nonmethylatable analog of cytidine, such as5-azacytadine. Other analogs will be known to those of skill in the art.Alternatively, such nonmethylatable analogs could be administered to asubject as drug therapy, alone or simultaneously with a sense structuralgene for TSLC1 or sense promoter for TSLC1 operably linked to TSLC1structural gene. Preferably the TSLC1 polynucleotide used in theinvention methods is derived from a mammalian organism, and mostpreferably from human.

Allelic loss of the TSLC1 locus (or LOH) can be detected by PCRamplification of polymorphic sequences flanking the TSLC1 gene. Both DNAfrom test cells and from healthy cells of the same individual are PCRamplified. The two PCR fragments representing both alleles are separatedon a polyacrylamide gel or by capillary electrophoresis. PCR productsderived from DNA of a healthy cells and of test cells are compared,wherein loss of one of two PCR fragments in test cells is indicative ofan allelic loss/genetic deletion of TSLC1 locus.

The present invention also provides a kit of parts for use in a methodof detecting HPV-induced invasive cancers and their precursor lesionsassociated with tumor suppressor lung cancer 1 (TSLC1) in test cells ofa subject. Such a kit may suitably comprise a brush or spatula to take a(cervical) scrape together with a container filled with collectionmedium to collect test cells. Alternatively, a sampling deviceconsisting of an irrigation syringe, a disposable female urine catheterand a container with irrigation fluid will be included to collectcervical cells by cervico-vaginal lavage.

A kit according to the present invention may comprise primers and probesfor the detection of TSLC1 promoter methylation, for the detection ofallele losses at chromosome 11q23.2 or for the detection of TSLC1 mRNAexpression. In another embodiment, a kit according to the invention maycomprise antibodies and reagents for the detection of TSLC1 proteinexpression in cervical scrapes or tissue specimens.

A kit of parts according to the invention comprises means for thedetection of TSLC1 promoter methylation or TSLC1 expression, such asTSLC1-specific antibodies, methylation-sensitive restriction enzymes, orprobes or primers capable of hybridising to the nucleotide sequence ofFIG. 1.

In yet another alternative embodiment of a kit of the invention themeans for the detection of TSLC1 promoter methylation or TSLC1expression may be combined with means for the detection of HPVinfection, preferably for the detection of HPV infection of thehigh-risk type. Such means may comprise HPV-specific primers or probes,protein markers for HPV infection or even surrogate markers for HPVinfection as are known in the art.

The present invention will now be illustrated by way of the following,non limiting examples.

EXAMPLES Example 1a Frequent TSLC1 Silencing in Cervical Carcinoma CellLines

TSLC1 mRNA expression levels were measured in normal cervical epithelialcells that were derived from biopsy samples, cervical smears and 11cervical carcinoma cell lines that contained high-risk HPV DNA. The mRNAlevels were measured by real time quantitative RT-PCR using Lightcyclertechnology (Roche) and compared with normal primary keratinocytes. Innormal cervical epithelial cells, the TSLC1 mRNA levels were comparableto those observed in primary keratinocytes. By contrast, TSLC1 mRNA wasundetectable in 7 of the 11 cell lines and severely reduced in another 3cell lines, showing that TSLC1 downregulation is apparent in 91% (10/11)of cervical carcinoma cell lines analysed.

On the other hand, TSLC1 expression was still abundant inHPV-immortalized cells. These HPV-immortalized cells are not yettumorigenic and have previously been shown to be representative ofpremalignant cervical lesions in vivo.

Subsequently, the analysis of the mechanisms underlying TSLC1downregulation was undertaken. Besides genetic events, i.e. deletionsand inactivating mutations, epigenetic events may result in genesilencing, as has been described for lung cancers [Kuramochi et al.,2001].

Example 1b Role of Methylation in TSLC1 Silencing in Cervical CancerCells

To assess whether a methylating event was underlying TSLC1 silencing incervical cancer cells, the cell lines SiHa, HeLa and CaSki were treatedwith the methylation inhibitor 5-aza-2′-deoxycytidine. TSLC1 mRNA levelswere compared to expression levels in primary epithelial cells, whichwas set to a 100%. Upon 5 to 7 days of incubation with5-aza-2′-deoxycytidine TSLC1 expression levels were upregulated from 0%to 26% in SiHa, and from 4% to 70% and 0% to 33% in HeLa and CaSkicells, respectively. These data indicate that in all these 3 cell linesTSLC1 downregulation results, at least in part, from a methylatingevent.

Next, TSLC1 promoter methylation by radioactive bisulfite sequencing wasstudied. It was found that all 6 CpG sites sequenced were methylated in9/11 cervical cancer cell lines. Except for three cell lines, no wildtype sequences were detected in these cell lines, indicating that TSLC1promoter methylation was clonal. In the remaining two cell lines none ofthe CpG sites were methylated. Except for one cell line, TSLC1 promotermethylation was correlated to reduced or undetectable TSLC1 mRNAexpression. No TSLC1 promoter methylation was detected in 4 isolates ofnormal epithelial cells and in the non-tumorigenic HPV-immortalizedcells.

Example 1c Role of a Chromosomal Deletion in TSLC1 Silencing

To analyse whether a chromosomal deletion attributed to TSLC1 silencingloss of heterozygosity (LOH) analysis was performed using 8 polymorphicmarkers flanking the TSLC1 gene. Normal DNA derived from eitherlymphoblasts or fibroblasts of 8 cervical carcinoma cell lines wasavailable. In 3 (38%) of these cell lines an allelic loss at 11q23.2 wasapparent. All 3 cell lines revealed TSLC1 promoter hypermethylation andabsence of detectable TSLC1 mRNA, suggesting that allelic deletioncombined with promoter hypermethylation underlied complete genesilencing.

Analysis on the HPV immortalized cell lines showed an LOH at 11q23.2 inall immortal passages of one of the four cell lines. However, TSLC1expression was still detectable in these passages and no promotermethylation was found, indicating that the retained allele is stillactively transcribed. No allelic loss at 11q23 was detected in the otherthree cell lines.

Taken together, these data show that TSLC1 silencing in cervical cancercells may result from 1) promoter methylation of one allele combinedwith deletion of the other allele as found in three of the seven celllines that displayed reduced TSLC1 expression, 2) promoter methylationwithout an allelic loss, suggesting that either both alleles arehypermethylated or that one is mutated, as was found in two cell lines,or 3) other yet unknown mechanisms, as was found in two cell lines.

Example 2 TSLC1 Suppresses Anchorage Independent Growth In Vitro andTumorigenicity in Nude Mice

Malignant transformation of hr-HPV infected epithelial cells in vitrohas been shown to proceed via the subsequent acquisition of 1) animmortal phenotype and 2) an anchorage independent phenotype, which canbe measured by the growth of cells in soft agarose.

We tested whether TSLC1 silencing was associated withanchorage-independent cell growth by comparing the growth in softagarose of TSLC1 expressing HPV-immortalized FK16A, FK16B, FK18A, andFK18B cells with that of the TSLC1 mRNA negative cervical cancer cellline SiHa cells. We found that the HPV-immortalized cells cultured insoft agarose produced either no colonies or a few colonies (0-100colonies per 5000 cells). By contrast, tumorigenic cervical carcinomaSiHa cells gave rise to approximately 700-800 colonies per 5000 cellswhen cultured in soft agarose.

To test the hypothesis that TSLC1 gene silencing was associated with theacquisition of the anchorage-independent growth phenotype, wetransfected SiHa cells with a TSLC1 expression vector or an emptycontrol vector, selected for cells stably transfected with each plasmid,and examined their TSLC1 mRNA expression, proliferation rate, andability to grow in soft agarose. All eight (100%) of the SiHa/TSLC1transfectants tested expressed TSLC1 mRNA and did not show an alteredproliferation rate compared with parental cells. However, seven (88%) ofthe eight SiHa/TSLC1 transfectants displayed a marked reduction inanchorage-independent growth compared with none of the four SiHa cellsbearing the empty vector (i.e., SiHa/hygro transfectants) anduntransfected SiHa cells (P=0.01) We tested four of the eight SiHa/TSLC1transfectants for tumorigenicity by injecting them into nude mice. All(7/7; 100%) injections with untransfected SiHa cells and SiHa hygrotransfectants, resulted in tumor volumes of at least 50 mm³ after 2 to 6weeks after injection compared with 0/8 (0%) injections with SiHa/TSLC1transfectants (P<0.001). In SiHa/TSLC1 transfectants tumor volumes of atleast 50 mm³ were seen at 7 to 12 weeks after injection or no tumorformation was seen._The delayed tumor growth of SiHa/TSLC1 transfectantsis most probably the result of the outgrowth of so called escaper cellsthat suppressed TSLC1.

In conclusion, these results show that TSLC1 can suppress both anchorageindependent and tumorigenic growth of cervical cancer cells, leavingimmortality and proliferation unaffected.

Example 3 Silencing of TSLC1 in Cervical Tissue Specimens

To determine whether and at what stage during cervical carcinogenesisTSLC1 silencing occurs in vivo, the methylation status of the TSLC1promoter was analyzed in cervical tissue specimens.

Since the tissue specimens consist of an admixture of normal stromalcomponents and abnormal cells the sensitivity of the assay to detectmethylated CpG islands in a background of normal unmethylated DNA wasdetermined. By analysing a dilution series of SiHa DNA (methylated TSLC1promoter) in DNA derived from primary keratinocytes (unmethylated TSLC1promoter), it was found that as low as 5% of methylated DNA in abackground of unmethylated DNA could still be detected with highreliability, using radioactive bisulfite sequencing on a Genomyx device.

Using this method none of 15 normal cervical epithelial biopsiesrevealed TSLC1 promoter methylation. Similarly, TSLC1 promotermethylation was undetectable in all low grade CIN lesions (n=12). Of 20high grade CIN lesions 35% showed methylation of the TSLC1 promoter. Inaddition, a total of 52 cervical squamous cell carcinoma sections wereanalysed, 58% (30/52) of which showed TSLC1 promoter methylation. Thus,TSLC1 promoter methylation appears to be a rather frequent event incervical squamous cell carcinomas and occurs late during the multistepsequence of carcinogenesis. Since in addition to TSLC1 promoterhypermethylation other alterations, including an allelic loss at11q23.2, may contribute to silencing of TSLC1 the actual percentage ofTSLC1 silencing in cervical carcinomas is likely to be even higher than58%.

Example 4 Detection of TSLC1 Promoter Methylation in Cervical Smears

In a small pilot study it was assessed whether the detection of TSLC1alterations can be applied to cervical smears and as such be used as amarker in cervical cancer screening programs.

For this, TSLC1 promoter methylation was analysed in archival smears ofwomen who developed cervical cancer. These scrapes were derived from aretrospective case-control which was designed to determine the value ofhr-HPV testing to signal false negative cervical smears in women whodeveloped cervical cancer and to assess whether hr-HPV is present innormal smears preceding cervical cancer (Zielinski et al., 2001; Br JCancer 85, 398-404). TSLC1 promoter hypermethylation was analysed inarchival index smears, taken from 11 women at the time of cervicalcancer diagnosis, as well as the corresponding cervical cancer biopsysamples available for 10 of these women. Six (6/11; 55%) of the indexsmears tested positive for TSLC1 promoter hypermethylation, as did thecorresponding cancer biopsy samples for those patients with an availablebiopsy sample.

As a follow up it was determined whether the detection of TSLC1 promotermethylation cannot only provide a diagnostic marker to detect invasivecervical cancer but may also provide a marker for risk assessment ofprogression to invasiveness. Five of the six patients that testedpositive for TSLC1 promoter hypermethylation, had archival cervicalsmears that were taken up to 19 years before cervical cancer wasdiagnosed and which were classified as either low grade squamousintraepithelial lesion (LSIL) or high grade intraepithelial lesion(HSIL). We found that smears that were taken up to 7 years prior tocervical cancer diagnosis had detectable levels of TSLC1 promoterhypermethylation, whereas smears taken more than 7 years prior tocervical cancer diagnosis tested had not. Moreover, we found that TSLC1promoter hypermethylation was mainly present in HSIL and rarely in LSIL,confirming that in cervical carcinogenesis the HSIL lesions are at thehighest risk of having invasive potential, but that also amongst LSILsthe lesions with invasive potential can be recognised.

No TSLC1 promoter methylation was detected in normal smears (n=15).

Example 5 TSLC1 Promoter Methylation Analysis by Methylation SpecificPCR (MSP)

To improve the sensitivity of detecting cells with TSLC1 promoterhypermethylation in a background of normal cells without TSLC1 promotermethylation we developed two novel methylation specific PCR tests. Thesetests are, similar to the bisulfite sequencing analysis as described inexamples 1b, 3 and 4, also based on bisulfite modification of DNA, inwhich the unmethylated cytosines are converted to an uracil whereas themethylated cytosines are protected from chemical modification. Each testconsists of one primer pair which specifically recognizes sequences withmethylated cytosines (i.e. unmodified cytosines following bisulfitetreatment) and a second primer pair which specifically recognizessequences with unmethylated cytosines (i.e. cytosine converted to uracilfollowing bisulfite treatment).

The first set of methylated and unmethylated DNA specific primer pairsspans nt −645 to −494 with respect to ATG (FIG. 1) for the methylatedprimer set and nt −646 to −496 (FIG. 1) for the unmethylated primer set.The second set of methylated and unmethylated DNA specific primer pairsspans nt −414 to 258 with respect to ATG (FIG. 1) for the methylatedprimer set and nt −414 to −254 (FIG. 1) for the unmethylated primer set.

To improve the specificity of the test the PCR products were hybridisedto internal methylated DNA and unmethylated DNA specific probes usingreverse line blot hybridisation. For this purpose the reverse primerswere labelled with biotine and hybridisation was essentially performedas described by van den Brule et al., J. Clin. Microbiol 2002. 40,779-787.

A third primer set was based on a MSP described by Jansen et al. (CancerBiol Ther. 2002 :293-6), spanning nt −695 to −582 with respect to ATG(FIG. 1) for the methylated primer set and nt −695 to −579 (FIG. 1) forthe unmethylated primer set. Also in this case the reverse primer wasbiotinylated and internal probes were selected for reverse line blothybridisation.

All MSPs were found to detect down to as low as 5 abnormal cells (i.e. 5cells with TSLC1 promoter hypermethylation) in a background of 1000normal cells (i.e. cells without TSLC1 promoter hypermethylation).

Using the combination of these MSPs in a pilot study TSLC1 promoterhypermethylation could be detected in 86% (12/14) of the cervicalcarcinomas analysed.

Example 6 Reduced TSLC1 Expression in Cervical Carcinomas

For the analysis whether the detection of TSLC1 promoterhypermethylation in cervical carcinomas is related to a reduced TSLC1protein expression the following experiment was performed.

Using anti-TSLC1 antibodies TSLC1 expression can be examined in thecourse of an immunohistochemical staining procedure as is known to thoseof skill in the art. In specimens immuno-stained with the TSLC1-specificantibody TSLC1 was detected in normal cervical epithelium, but notdetectable in cervical carcinoma cells. This fact is caused by TSLC1promoter hypermethylation effecting reduced TSLC1 protein levels withinthe invasive carcinoma cells.

In summary, TSLC1 gene silencing is a highly frequent event inHPV-associated cervical carcinomas. It appears far more frequent than incarcinomas which are not associated with an HPV-infection and which havea different etiology. For example TSLC1 silencing has been detected in40% of lung cancers (Kuramochi et al., 2001; Nature Genet 27, 427-430)33% of breast cancers [Allinen et al.,], 32% of prostate cancers[Fukuhara et al., 2002], 27% of primary pancreatic adenocarcinomas[Jansen et al., 2002] and 34% of nasopharyngeal carcinoma (Bik-Yu Hui etal., 2003; Mol Carcinogenesis, 38, 170-178).

Moreover, in vitro studies revealed a functional involvement of TSLC1silencing in progression to tumorigenicity of an HPV-transformed cell.It is likely that the absence of TSLC1 protein expression and/or thepresence of TSLC1 promoter hypermethylation is not only a biomarker forthe progression to invasiveness of a premalignant HPV-infected lesionbut also for prognosis of invasive cervical cancers. In adenocarcinomasof the lung a reduced TSLC1 expression has indeed been associated with apoor prognosis (Uchino et al., Cancer, 2003, 98, 1002-1007).

Example 7 Reduced Expression of TSLC1 and/or TSLC1 PromoterHypermethylation in HPV-Induced Carcinomas of the Oropharynx and theirPrecursor Lesions

It can be expected that in the group of oropharyngeal carcinomas causedby HPV the TSLC1 promoter is hypermethylated and TSLC1 proteinexpression is absent or reduced in an identical way as described abovefor cervical carcinomas. Hence, for the analysis of HPV-associatedoropharyngeal carcinomas and their precursor lesions an experiment asfor example the following may be performed. Using anti-TSLC1 antibodiesTSLC1 expression can be examined in the course of an immunohistochemicalstaining procedure as is known to those of skill in the art. Similarly,TSLC1 promoter hypermethylation can be examined on the same specimen bybisulfite sequencing as described in Example 3 or MSP procedures asdescribed in Example 5. In oropharyngeal cancer specimens and specimensof their precursor lesions with invasive potential and consequentlymetastatic potential immuno-staining with anti-TSLC1 antibodies mayyield a markedly reduced signal, compared to normal oropharyngealepithelium. Moreover, the specimens with reduced immuno-staining mayreveal TSLC1 promoter hypermethylation. Prediction of the invasive andmetastatic potential may have major influence on individual treatmentstrategies of patients with HPV-associated precursor lesions oforopharyngeal cancer or invasive oropharyngeal cancer.

1. A method of detecting art HPV-induced invasive cancer or a precursorlesion thereof associated with tumor suppressor lung cancer 1 (TSLC1) ina subject, the method comprising contacting a target cellular nucleicacid component of in a test cell with a reagent that detects TSLC1, anddetecting a reduction in the TSLC1 in the test cell as compared to thatof a comparable normal cell, detecting an increase or decrease inmethylation of the TSCL1 promoter in the test cell, as compared to acomparable normal cell, or both.
 2. A method according to claim 1,wherein the target cellular component is a nucleic acid.
 3. A methodaccording to claim 2, wherein the nucleic acid is mRNA.
 4. A methodaccording to claim 1, wherein the reagent is a nucleic acid probe orprimer that binds to TSLC1.
 5. A method according to claim 1, whereinthe subject has loss of heterozygosity at chromosome 11q23.